The Vif protein is highly conserved among lentiviruses, and is crucial for HIV-1 replication in primary T lymphocytes and macrophages, the cells infected by the virus in vivo. Our work has demonstrated that Vif is necessary at the time of viral particle formation, to make HIV-1 competent for proviral DNA synthesis. Furthermore, our recent analyses indicate that Vif stabilizes the HIV-1 reverse transcription complex in vitro. Progress in the Vif field has been slowed down by the technical difficulty of obtaining high-titer stocks of functionally relevant virus. We will use two parallel approaches to solve this problem. This will facilitate the further exploration of VIF function and mechanisms of action, using a combination of functional and biochemical analyses. Finally, the search for viral and cellular proteins interacting with Vif will complement these studies. our specific aims are thus as follows: 1) Creation of regulated vif expression systems, utilizing a tetracycline- repressible and an ecdysone-inducible system. 2) Generation of conditional vif mutants, by site-directed and saturation mutagenesis. 3) Continuing studies of Vif function and mechanism of action, by further analyzing the Vif-mediated enhancement of reverse transcription, studying the processing and post-translational modification of virion-associated proteins, and examining the early steps of infection through new protocols developed in our laboratory. 4) Search for viral and/or cellular proteins that associate with Vif, by co-immunoprecipitation and using the yeast two-hybrid system.